Tissue stem cells provide for the maintenance and regeneration of organs and tissues throughout life, and the ability of stem cells to contribute to tissue homeostasis depends on their unique ability to generate new stem cells (self-renewal), as well as specialized cell types (differentiation). Stem cells are found in discrete, specialized microenvironments (niches) where they are exposed to diverse systemic and local signals that are integrated with cell intrinsic factors to regulate stem cell behavior. Numerous intrinsic and extrinsic changes occur with age that result in altered stem cell behavior, leading to reduced tissue maintenance and regeneration. This lab has developed Drosophila melanogaster as a system to probe the effects of aging on stem cells and the niche. In the Drosophila testis, stem cells surround and contact the apical hub, a cluster of somatic cells that express the self-renewal factor Unpaired (Upd), which activates the JAK-STAT pathway in adjacent stem cells to specify self-renewal and maintenance. In previous years of this grant, it was shown that aging results in a decrease in upd expression in hub cells. Recent work by the lab has revealed that IGF-II mRNA binding protein (Imp) counteracts endo siRNAs to stabilize upd RNA in hub cells. However, Imp expression decreases in hub cells of older males, similar to upd, which is due to targeting of Imp by the heterochronic microRNA let-7. Therefore, in the absence of Imp, upd mRNA becomes unprotected and susceptible to degradation, leading age-related loss of niche function. Imp and let-7 are also expressed in germ cells, albeit at lower levels, and let-7 is expressed in the midgut of young, but not old, animals. Therefore, this proposal will build upon previous findings by addressing the following Specific Aims: Specific Aim 1) To characterize the role of Imp in germ cells and germline aging. Specific Aim 2) To characterize a role for let-7 in the intestinal stem cell niche. Specific Aim 3) To uncover the mechanism by which let-7 expression changes during aging in the testis and intestine. Reports have indicated that the let-7 family of miRNAs likely target mammalian Imp homologs (Igf2bps1-3). Given the broad role of the let-7 family in aging, stem cells, cancer and metabolism, the regulation of Imp by let-7 is likely an important, conserved mechanism in numerous physiological processes. In addition to providing insights into tissue homeostasis, studies focusing on age-related changes in stem cells and their specialized microenvironments will help to identify and overcome unique hurdles in the manipulation of tissue stem cells derived from older patients and to facilitate the development of techniques for regenerative medicine to treat aging-related diseases.